Smad proteins transduce the TGF-ß family signal at the cell surface into gene regulation in the nucleus. In addition to being phosphorylated by the TGF-ß family receptors, Smads are phosphorylated by a variety of intracellular kinases. The most studied are by cyclin-dependent kinases, the MAP kinase family members, and GSK-3. Phosphorylation by these kinases regulates Smad activities, leading to various biological effects. This chapter describes the methods for analyzing Smad phosphorylation by these kinases.